Increasing energy costs and environmental concerns have emphasized the need to produce sustainable renewable fuels and chemicals. Fatty acids are composed of long alkyl chains and represent nature's “petroleum,” being a primary metabolite used by cells for both chemical and energy storage functions. These energy-rich molecules are today isolated from plant and animal oils for a diverse set of products ranging from fuels to oleochemicals.
Whereas microbial fermentation processes for producing ethanol and related alcohol biofuels are well established, biodiesel (methylesters of fatty acids) is the major long chain product produced biologically, and it is almost exclusively derived from plant oils today. However, slow cycle times for engineering oil seed metabolism and the excessive accumulation of glycerol as a byproduct are two major drawbacks of deriving biodiesel from plants.
Although most bacteria do produce fatty acids as cell envelope precursors, the biosynthesis of fatty acids is tightly regulated at multiple levels and large quantities are not made. Thus, the production of fatty acids from bacteria has not yet reached the point where it is cost effective.
Our laboratory has already made considerable progress in engineering bacteria to produce more free fatty acids than are normally found in native bacteria. WO2011116279 for example, describes a recombinant bacterium comprising at least one overexpressed acyl-ACP thioesterase gene, and wherein at least one gene from the tricarboxylic acid cycle or glycolysis or both is inactivated to drive carbon in the direction of fat production. For example, an ACP thioesterase was combined with deletions in native fadD, and sucC. These bacteria have significantly increased overall fat levels.
WO2013096665 describes the next step in our work, which was to engineer a microorganism for producing enhanced amounts of long chain fatty acids, having an overexpressed acyl ACP thioesterase, and at least one mutated gene selected from the group consisting of fabR, fabZ, fadR, fabH and combinations thereof, and optionally including a inactivated sucC gene. Various bacteria in this category produce more long chain fats.
The next step was to enable the production of odd chain length fatty acids. Odd chain fatty acids can be made as described in US20140193867. In that application, the starting material was manipulated to be a C3 molecule, propionyl-CoA, by overexpressing a propionyl-CoA synthase gene. We also replaced the native β-ketoacyl-acyl carrier protein synthase III gene with one having a greater substrate preference for propionyl-coA than acetyl-coA. With these three modifications, greater odd chain fats were produced that was heretofore possible. In fact, >80% of the fats produced by such strains were of odd chain lengths.
The above genetic manipulations provided significant improvements in fat levels, and the excretion of visible amounts of fats also provided an easy method of collecting fats, while keeping the culture active and undisturbed, churning out more fats. Further, the addition of Mg+2 to the culture allowed improved production as well.
Another improvement would be able to make hydroxyl- or dicarboxylated fatty acids. Hydroxy fatty acids widely used for making polymers are also valuable in chemical, cosmetic and food industries as starting materials for synthesis of lubricants, adhesives, and cosmetic ingredients. Similarly, dicarboxylic fatty acids have many industrial applications such as synthesis of copolymers like polyamides and polyesters, coatings, adhesives, greases, polyesters, dyestuffs, detergents, flame retardants, cosmetic ingredients, and fragrances. For example, adipic acid (n=6) is among the top 50 bulk manufactured chemicals in US primarily used for manufacturing nylon. Sebacic acid (n=8) and its derivatives have many applications used in manufacturing plasticizers, lubricants, and cosmetics. Dodecanedioic acid (n=12) is used in the production of nylon (nylon-6,12) and polyamides.
WO2013024114 describes the microbial preparation of co-functional carboxylic acids and carboxylic acid-functionalized co-esters. However, this method is not dependent on fatty acids as starting materials. Further, only a single experiment was performed, and the yields were quite poor.
This disclosure addresses some of those improvements.